The present invention is concerned with methods and apparatus for mass spectrometers, especially TOF (“time-of-flight”) mass spectrometers. In particular, the present invention relates to methods and apparatus for ion axial spatial distribution focusing.
TOF mass spectrometry is an analytical technique for measuring the mass to charge ratio of ions by accelerating ions and measuring their time-of-flight to a detector.
Two known methods of TOF mass spectrometry are matrix-assisted laser desorption/ionization TOF mass spectrometry (“MALDI TOF” mass spectrometry) and tandem TOF mass spectrometry (“TOF-MS/MS” mass spectrometry). Maldi TOF-MS and TOF-MS/MS are long established as methods of identifying macro-molecular compounds in biological systems for example.
In Maldi TOF-MS a laser pulse is focussed to a small spot (“laser spot”) on a mixture of a sample of the biological material and a light-absorbing matrix on a sample plate so that a pulse of ions is produced.
This ion pulse is analysed and detected with a time-of-flight mass spectrometer, TOF-MS, so that the mass to charge ratio of the ions is measured.
In TOF-MS/MS mass spectrometry, ions undergo fragmentation before they are analysed and detected. The ions may be fragmented by meta-stable decay (post-source decay, PSD) or by collision induced dissociation (CID), for example. TOF-MS/MS is useful because it allows analysis of both precursor ions (non-fragmented ions) and product ions (fragmented ions). TOF-MS/MS mass spectrometry can be used in combination with MALDI TOF mass spectrometry. In other words, a MALDI ion source can be used in a mass spectrometer in which ions undergo fragmentation before they are detected.
In the ion source of the TOF mass spectrometer, at the time of extraction of the ions there are different distributions of the ions that characterise their initial direction, position and energy. For example, the range of radial position (distance from the ion optical axis) is determined by the spot size, as illustrated in FIG. 1. Thus, after desorption from the sample plate 1, ions 2, 4 are spaced from the ion optical axis 6 (the main axis of the spectrometer) by a distance R. In the case of a MALDI source the size of R is dictated by the diameter of the “laser spot”, being the area from which ions are generated from the sample by the laser beam.
Each point in the ion source can generate a distribution in the initial direction or at an angle to the ion optical axis, as illustrated in FIG. 2. Thus, an ion 10 may possess a velocity having a radial component that is such as to cause them to travel away from the source at an angle θ to the ion optical axis 6. This characterises the expansion of the ion plume 12 outwards from the centre of the spot 14.
The ions are also produced with a range of initial energy or speed, as illustrated in FIG. 3. Thus, ions 20, 22 within the ion plume have different energy or velocity, such that, for example, the energy E1 of ion 20 may be smaller than the energy E2 of ion 22.
In the case of a Maldi sample the axial velocity distribution corresponds to a distribution in what is commonly known as the Jet velocity, typically around a few hundred ms−1.
There is also a spatial distribution of ions in the axial direction normal to the sample surface, as illustrated in FIG. 4. This can be due to the different starting positions of ions because of sample topography and/or thickness. It can also be due to different starting times for ions coupled with the axial velocity. Thus, ions 30, 32 are separated in space in the axial direction (being parallel to the ion optical axis 6) by a distance Z.
Each of the distributions affects the performance of the TOF-MS (and TOF-MS/MS) and the result is measured by the width of the peak for a single mass to charge which in turn determines the mass resolution.
The size of the effects can be controlled by various means. For example, the radial spatial distribution is set by the size of the focussed laser spot and controlled by the collimation of the ion optical lenses in the mass spectrometer. Similarly, the effect of the angular distribution is also controlled by the lenses in the ion optics.
It is possible to compensate for either the axial spatial distribution or the velocity distribution with pulsed extraction by using the spatial distribution of the ions in combination with the pulsed electrostatic field to produce a space focus in the flight tube [Time-of-Flight Mass Spectrometer with Improved Resolution’ W. C. Wiley and I. H. McLaren, Rev. Sci. Instrum., 26, 1150 (1955)]. The space focus is a point where all the ions in the velocity distribution come together at the same time. The space focus can be at the detector in the case of a linear time of flight or it can be the front focus of the ion mirror in a reflectron time of flight.
However, in using pulsed extraction from the ion source, it is well known that only one axial distribution can be focussed at a time. Either the initial axial spatial distribution or the initial axial velocity distribution can be brought to a space focus but not both distributions simultaneously.
In the case of orthogonal extraction of ions from a beam such as in electrospray TOF-MS, the axial spatial distribution is focussed by pulsed extraction whilst the axial velocity distribution for the time of flight is negligible being in the orthogonal direction.
In an ion source such as a MALDI or SIMS ion source, the ions are desorbed from the surface of a sample deposited on a plate by using pulsed extraction, which focuses the velocity distribution. This works on the basis that the size of the initial axial spatial distribution is much less than the spatial distribution of the ions produced by the velocity distribution during the delay time before pulsed extraction. However, this will only be the case if the sample is very thin (a few microns) and/or if the laser power is very close to the threshold for generating ions so that they originate only from the surface of the sample.
Thus, using an appropriate design of ion optics to collimate the ion beam coupled with a pulsed extraction ion source where the depth of desorbed sample is very thin and the laser power is very close to the threshold, it is possible to achieve very high mass resolutions for TOF-MS.
In TOF-MS the ions extracted from the ion source arrive at the detector intact so that the mass to charge ratio of the molecules from the sample is measured. If the ions are made to break up into smaller pieces or fragments, in the field free region, it is possible with a reflectron TOF-MS to measure the mass to charge ratio of the fragment ions and so carry out TOF-MS/MS. This technique, also known as tandem TOF or TOF/TOF allows the analysis of the structure of the molecules desorbed from the sample. So, for example, the amino-acid sequence of a peptide or protein sample can be determined from the TOF-MS/MS or fragment spectrum.
In TOF-MS/MS the ions fragment in the field free region of the TOF and do so either by the process of metastable decay and/or by collision with a neutral gas in a region of high pressure (CID).
When ions fragment in a field free region such as the flight tube or collision cell, where there are no external forces, the fragments continue with the velocity that is effectively the same as that of the parent (pre-cursor) ion. This in turn means that the energy of the fragment ion is reduced to the fraction of the parent ion energy in the ratio of the fragment mass to the parent mass. In other words, the following relationship applies, where Ef is the kinetic energy of the fragment ion, Ep is the kinetic energy of the parent ion, mf is the mass of the fragment ion and mp the mass of the parent ion:Ef=Ep·mf/mp 
With a linear TOF-MS, there is no way of distinguishing between the fragment ions and the parent (pre-cursor) ions because they have the same velocity and therefore same flight time to the detector. However, as noted above, it is possible to distinguish between fragment ions by using a reflectron. A reflectron is effectively an energy analyser because the distance traveled by the ions into a reflectron is determined by the point at which the electrostatic potential is equal to the kinetic energy of the ions as they enter the reflectron. For fragment ions the distance traveled into the reflectron is a function of the energy which is determined by the ratio of the fragment mass to the parent mass. Since the flight time through the reflectron is dependent on the distance traveled into the reflectron, the time-of-flight of the fragment ion becomes a function of the ratio of the fragment mass to the parent mass.
In principle therefore any reflectron is capable of producing a TOF-MS/MS spectrum. However, because the parent ions have an initial energy distribution the fragment ions also have an energy distribution. The relationship between the nominal ion energy and the distance from the reflectron to the detector at which ions with differing initial energies are focussed, depends on the shape of the field or voltage distribution in the reflectron. The most common reflectrons have voltage distributions which vary linearly from the front to the back. Often (to make them more compact) there are two or more sections in one reflectron, each with different voltage gradients. For such linear field reflectrons the distance between the reflectron and the appropriate location of the detector also varies linearly with the nominal ion energy. It follows that the position of the detector for optimum mass resolution will vary linearly with fragment mass. However, in practice, because the detector is a fixed distance from the reflectron, the mass resolution for fragments falls rapidly as the mass reduces from the parent mass. The result is that a linear field reflectron cannot on its own produce a TOF-MS/MS spectrum where the complete fragment mass range is in focus and has good mass resolution.
Early instruments got around this problem by stepping the reflectron voltage and analysing a small segment of the fragment spectrum at a time. The major disadvantage of this was the need to collect multiple spectra and then ‘stitch’ them together which results in long experiment times and high sample consumption.
Recently, manufacturers have got around this problem by re-accelerating the ions after the point where fragmentation has taken place so that the range of fragment energy is effectively compressed into the narrow range for which good mass resolution is produce by the linear reflectron. So called TOF/TOF instruments [see for example U.S. Pat. No. 6,512,225 (Vestal) and U.S. Pat. No. 6,703,608 (Holle)] either start with, or slow down the ions to, a low energy typically 1 keV to 8 keV and then re-accelerate them by means of a second pulsed extraction region to a nominal energy around or greater than 20 keV. Such instruments have the disadvantages of being complex and expensive because of the additional pulsed high voltage fields required.
An alternative method is to use a reflectron where the potential distribution is non-linear so that the range of distance to the detector for different fragment ion mass is much smaller than for a linear reflectron. Such a reflectron is known as the curved field reflectron as described in U.S. Pat. No. 5,464,985 (Cotter). In this case it is possible to measure a complete TOF-MS/MS spectrum with good fragment ion mass resolution without re-acceleration of the fragment ions. Ions therefore have nominal energies of 20 keV from the source through to the reflectron. This method has the advantages of lower complexity and cost but also allows higher initial energies and therefore higher collision energies if using CID. However, one disadvantage is that the best mass resolution that can be achieved for the fragment ions is not as high as those instruments in which re-acceleration is used.
In the case of fragment ions produced by metastable decay (post-source decay, PSD), the production of the fragment ions relies on excess internal energy in the pre-cursor ions to cause the pre-cursor ions to fragment. The extra energy is produced in a MALDI ion source by increasing the laser fluence to well above the threshold required for ion generation.
In the case of collision induced dissociation (CID) the fragmentation is caused by high energy collisions with the neutral gas molecules. However, for efficient CID from a MALDI ion source, the laser power still has to be above the threshold level.
A consequence of the extra laser power required for TOF-MS/MS is that the mass resolution of the pre-cursor ions, and therefore also of the fragment ions, is much lower than for TOF-MS where the laser power is close to the threshold.
U.S. Pat. No. 5,739,529 (Laukien) describes a method for compensating the axial spatial distribution in reflectron TOF-MS. There, a pulsed electrostatic field is applied using electrodes located either in the reflectron or between the reflectron and the detector to focus the spatial distribution at the detector. This method provides for an improvement in mass resolution for TOF-MS ions over a very narrow mass range.
However, the present inventors have noted that this method is not suitable to compensate the spatial distribution for TOF-MS/MS because the fragment ions are separated in time by the reflectron so that only a narrow mass range of fragments could be focussed.
The present invention seeks to address this and other drawbacks associated with known methods of performing TOF-MS/MS described above.